Immunohistochemistry (IHC) is widely known as a histological (histochemical) technique in which an antigen in a tissue sample is detected using an antibody. This immunohistochemistry may be referred to as “immunostaining” or the like, from the standpoint of carrying out a color-developing process in order to visualize antigen-antibody reaction which is originally invisible (hereinafter, in the present specification, the term “immunohistochemical staining” may be also used for this immunohistochemistry). Due to its feature of visualizing the location where the antigen-antibody reaction is, the immunohistochemistry is widely used in the fields of medicine and life chemistry for the purpose of detecting the location where a biological substance in a tissue sample is.
In addition, as a histological (histochemical) technique related to the immunohistochemistry, lectin staining is also known. This lectin staining is a technique for detecting a sugar chain in a tissue sample using lectin by using the nature of lectin nonimmunologically and specifically binding to a particular sugar chain, and is widely used in the fields related to sugar chains.
In an IHC method, a staining method that allows bright field observation is widely used as a method for visualizing the location where the antigen-antibody reaction is. Specifically, a technique that uses a substrate to be converted into a pigmentary substance by an enzyme is commonly used. For example, in clinical sites, a method, in which an anti-HER2 antibody bound to a HER2 antigen site in a tissue sample is stained and visualized using diaminobenzidine (DAB), and the expression amount of the HER2 is detected by bright field observation via the visualized anti-HER2 antibody, is widely carried out. This bright field observation has the advantage that, by comprehensively judging from the information obtained in an analog manner which is about the color and so on resulting from the staining, more detailed information on the target molecule can be obtained as compared to the method using a light-emitting substance as described below.
However, there is a problem with the quantitativity of its results, since, in a staining method using an enzyme reaction like the DAB method, the stainability is changed depending on the staining conditions including the reaction time (see, for example, Non-patent Documents 1 and 2). In addition, its criteria lack quantitativity because of their roughness since the staining level is graded in only 4 scores of from 0 to 3, and further the judgment using the criteria depends on the level of skill of the pathologist or the like, which have been problematic.
In the IHC method, a visualization method that uses a substance having a color-developing property in itself (hereinafter also referred to as a “light-emitting substance”) is also used, and, for example, phosphors are suitably used therein (immunostaining that uses a phosphor is hereinafter also referred to as “immunofluorescence staining”). According to the visualization technique using a phosphor, quantitative evaluation can be carried out, for the technique has an excellent reproducibility since the change in the stainability depending on the staining conditions including the reaction time of the enzyme is small, and, the location and the expression amount of a biological substance can be digitally analyzed (see, for example, Non-patent Document 2).
However, in clinical sites, the staining methods using DAB or the like which allows bright field observation as described above have been used for many years, and know-how for evaluating the pathology has been accumulated. Consequently, a skilled pathologist could judge the pathology more rightly and in more detail on the basis of information about the color and so on resulting from the staining, as compared to the case of a method using a light-emitting substance.
Accordingly, there is a problem that, in the case of using only the information obtained by visualization using a light-emitting substance such as a phosphor, quantitativity can be expected but the more right and detailed pathology judgment based on the experiences of the skilled pathologists can not be expected.
In addition, as a method of examination in which a biological substance in a tissue sample is quantitatively detected, fluorescence in situ hybridization method (FISH method), in which gene expression amount is evaluated, is also used in clinical sites. For example, a method, in which a probe for detecting the HER2 gene and a probe for detecting the centromere of chromosome 17 are used; the HER2 gene copy number per chromosome 17 is counted; and, based on the results thereof, whether the HER2 gene has been amplified or not is judged, is carried out.
However, the FISH method is a quantitative examination method, but is not a method by which the expression amount and/or the intracellular localization of a protein are directly evaluated, which has been problematic.